P2X受体,由细胞外ATP激活的配体门控离子通道亚家族,与各种病理生理过程有关,包括炎症,疼痛感知,以及免疫和呼吸调节。使用晶体学和低温EM进行的结构测定表明,不同物种之间不同P2X亚型的细胞外三维结构非常相同,大大推进了我们对P2X激活机制的理解。然而,结构研究产生了不同亚型的胞内结构域(ICD)的矛盾结构(例如,P2X3和P2X7)处于apo状态,ICD在P2X功能调节中的作用尚不清楚.这里,我们认为P2X3受体的ICD具有类似于开放状态的apo状态构象,但具有较少紧张的结构,含有影响P2X3生理和病理作用的变构位点。使用共价占据,工程二硫键和电压钳荧光法,我们建议ICD可以与P2X3的跨膜结构域进行协调运动,从而促进通道激活。此外,我们鉴定了一种新的P2X3增强子,PSFL77,并揭示了其位于ICD1α3β结构域的潜在变构位点。PSFL77调节P2rx3+/+的疼痛感知,但不是在P2rx3-/-中,老鼠,表明1α3β,与P2X3功能调节有关的“可调”区域。因此,当P2X3处于apo状态时,它的ICD架构相当有序,而不是非结构化的向外折叠,实现P2X3受体信号的变构调节。
P2X receptors, a subfamily of ligand-gated ion channels activated by extracellular ATP, are implicated in various physiopathological processes, including inflammation, pain perception, and immune and respiratory regulations. Structural determinations using crystallography and cryo-EM have revealed that the extracellular three-dimensional architectures of different P2X subtypes across various species are remarkably identical, greatly advancing our understanding of P2X activation mechanisms. However, structural studies yield paradoxical architectures of the intracellular domain (ICD) of different subtypes (e.g., P2X3 and P2X7) at the apo state, and the role of the ICD in P2X functional regulation remains unclear. Here, we propose that the P2X3 receptor\'s ICD has an apo state conformation similar to the open state but with a less tense architecture, containing allosteric sites that influence P2X3\'s physiological and pathological roles. Using covalent occupancy, engineered disulfide bonds and voltage-clamp fluorometry, we suggested that the ICD can undergo coordinated motions with the transmembrane domain of P2X3, thereby facilitating channel activation. Additionally, we identified a novel P2X3 enhancer, PSFL77, and uncovered its potential allosteric site located in the 1α3β domain of the ICD. PSFL77 modulated pain perception in P2rx3+/+, but not in P2rx3-/-, mice, indicating that the 1α3β, a \"tunable\" region implicated in the regulation of P2X3 functions. Thus, when P2X3 is in its apo state, its ICD architecture is fairly ordered rather than an unstructured outward folding, enabling allosteric modulation of the signaling of P2X3 receptors.